Hispidulin inhibits adipogenesis in 3T3-L1 adipocytes through PPARγ pathway

Accepted Manuscript Hispidulin inhibits adipogenesis in 3T3-L1 adipocytes through PPARγ pathway Seul Gi Lee, Jin Soo Kim, Kyoungjin Min, Taeg Kyu Kwon, Ju-Ock Nam PII:

S0009-2797(17)31259-0

DOI:

10.1016/j.cbi.2018.07.027

Reference:

CBI 8372

To appear in:

Chemico-Biological Interactions

Received Date: 20 November 2017 Revised Date:

25 June 2018

Accepted Date: 24 July 2018

Please cite this article as: S.G. Lee, J.S. Kim, K. Min, T.K. Kwon, J.-O. Nam, Hispidulin inhibits adipogenesis in 3T3-L1 adipocytes through PPARγ pathway, Chemico-Biological Interactions (2018), doi: 10.1016/j.cbi.2018.07.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

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preadipocyte

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Hispidulin

Differentiating adipocyte

Mature adipocyte

ACCEPTED MANUSCRIPT Hispidulin inhibits adipogenesis in 3T3-L1 adipocytes through PPARγ pathway

a

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Seul Gi Lee a, Jin Soo Kim a, Kyoungjin Min b, Taeg Kyu Kwon b,and Ju-Ock Nam a c*

Department of Food Science and Biotechnology, Kyungpook National University,

b

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Daegu 41566, Republic of Korea

Department of Immunology, School of Medicine, Keimyung University, 2800

c

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Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, Republic of Korea

Institute of Agricultural Science & Technology, Kyungpook National University,

Daegu 41566, Republic of Korea

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*Correspondence to: Ju-Ock Nam

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Tel: +82-53-950-7760, Fax: +82-53-950-7762, E-mail address: [email protected]

ACCEPTED MANUSCRIPT Abstract Hispidulin, a natural flavone, has been reported to have diverse pharmacological effects, including antifungal, antioxidant, and antithrombotic properties. However, an anti-

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adipogenic effect has not yet been reported, which is the focus of the current study. Hispidulin suppressed the differentiation of adipocytes and cellular lipid accumulation without cytotoxicity. Treatment with hispidulin at concentrations of 10, 20, and 40 µM

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reduced intracellular lipids by 88.1%, 81.9%, and 75.8%, respectively. In addition, hispidulin reduced mRNA and protein expression of peroxisome proliferator-activated

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receptor gamma (PPARγ) and adiponectin. To our knowledge, these results are the first evidence of the anti-adipogenic effects of hispidulin in 3T3-L1 adipocytes, indicating

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that hispidulin has potential as a novel anti-obesity therapeutic.

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Keywords: 3T3-L1 adipocytes, anti-adipogenic, hispidulin, PPARγ, sage

1. Introduction

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Obesity, a serious public health problem, is caused by excess energy intake and lack of energy expenditure [1, 2]. The disruption of energy homeostasis results in abnormal adipocyte differentiation, which is characterised by hyperplasia (increased number) and hypertrophy (increased size) of adipocytes [3]. The 3T3-L1 cell line is well-established as a good model for studying the in vitro adipogenic differentiation process that is related to obesity. 1

ACCEPTED MANUSCRIPT Hispidulin (4′,5,7-trihydroxy-6-methoxyflavone) is a natural phytocompound, extracted from various plants, such as sage and thistle [4, 5]. A previous study demonstrated that an extract of Salvia plebeia R. Br. containing hispidulin had anti-

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obesity effects in vivo [6]. However, S. plebeia extract contains many flavonoids, polyphenols, and saponins, in addition to hispidulin [6]. Therefore, it is unclear whether hispidulin alone is effective to prevent or treat obesity. In addition, there have been

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several studies reporting that hispidulin has antifungal, antiproliferative, antioxidant, and antithrombotic properties [7, 8]. However, its anti-obesity effects were not

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investigated.

In the present study, we demonstrate that hispidulin inhibits the differentiation of 3T3L1 adipocytes by downregulating the expression of key inducers of adipogenesis, namely

peroxisome

proliferator-activated

receptor

gamma

(PPARγ)

and

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CCAAT/enhancer binding protein alpha (C/EBPα).

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2. Materials and Method

2.1. Chemicals and cell culture Hispidulin and hydroxycitric acid (HCA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and chromade (Santa Ana, CA, USA), respectively. 3T3-L1 preadipocytes were obtained from the Korean Cell Line Bank (KCLB) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% newborn calf serum (BCS) at 37°C in a humidified 5% CO2 2

ACCEPTED MANUSCRIPT incubator. For differentiation into mature adipocytes, 3T3-L1 preadipocytes were treated with adipogenic cocktail (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 µM dexamethasone (DEX), 167 nM insulin, and 100 µM indomethacin) in DMEM

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containing 10% foetal bovine serum (FBS) for 48 h. Afterward, stimulated cells were maintained in medium supplemented with 10% FBS and 10 µg/mL insulin for 5 days. To examine whether hispidulin treatmentinhibits differentiation, 3T3-L1 cells were

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treated with hispidulin or HCA (used as a positive control drug) during the differentiation period. The preadipocytes and mature adipocytes treated with the same

control (PC), respectively.

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2.2. Cytotoxicity assay

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amount of DMSO vehicle were considered as the negative control (NC) and positive

3T3-L1 preadipocytes were seeded into 96-well plates and maintained overnight. Afterward, cells were treated with hispidulin and HCA at concentrations of 0 40 µM for

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24 h or were treated with 0.1~1.6 ul DMSO, as a control group (CON). MTT (3-(4,5)dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) solution was added to each well

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and incubated for 3 h at 37ºC, and the reaction products were dissolved with isopropyl alcohol (Duksan Pure Chemicals, Korea). The absorbance of each sample was measured at 595 nm.

2.3. Oil Red O staining (ORO)

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ACCEPTED MANUSCRIPT Oil Red O staining (ORO) was performed as previously described [1]. Briefly, preadipocytes and differentiated adipocytes were washed, fixed, and stained with Oil Red O solution (Sigma-Aldrich, St. Louis, MO, USA). Then, cells were washed with

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distilled water and photographed with a microscope at 200× magnification. To quantify the level of staining, the Oil Red O stain in the cells was dissolved in isopropyl alcohol

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and its absorbance was measured at 495 nm.

2.4. Triglyceride assay

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The triglyceride (TG) assay was performed as previously described [1]. Briefly, preadipocytes and differentiated adipocytes were lysed in 5% NP-40 lysis buffer and TG content was measured using a TG Quantification Kit (Bio Vision, California, USA)

measured at 570 nm.

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according to the manufacturer’s instructions. The absorbance of each sample was

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2.5. Real-time reverse transcription polymerase chain reaction (RT-PCR)

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Total RNA was extracted by the TRIzol® method, according to the protocol recommended by the manufacturer (TaKaRa Bio Inc., Japan). Complimentary DNA (cDNA) was synthesized using the PrimeScript™ RT Reagent Kit (TaKaRa Bio). Quantitative PCR was performed with iCycler iQ™ Real-Time PCR Detection System (Bio-Rad Laboratories, USA) using SYBR Green (TOYOBO, Japan). The sequences of primers

were

as

follows:

mouse

GGAAGACCACTCGCATTCCTT-3’;

PPARγ

(AB644275.1) reverse,

(forward,

5’5’4

ACCEPTED MANUSCRIPT GTAATCAGCAACCATTGGGTCA-3’), C/EBPα (NM_001287523.1) (forward, 5’CAAGAACAGCAACGAGTACCG-3’; GTCACTGGTCAACTCCAGCAC),

adiponectin

reverse, (NM_009605.4)

5’(forward,

5’-

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GATGGCACTCCTGGAGAGAA-3’; reverse, 5’-TCTCCAGGCTCTCCTTTCCT-3’) and β-actin (NM_007393.4) (forward, 5’- CGTGCGTGACATCAAAGAGAA-3’; reverse, 5’-GCTCGTTGCCAATAGTGATGA-3’). All reactions were performed in

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triplicate.

2.6. Western blotting

Cells lysates and western blot analyses were performed as previously described [9]. Briefly, cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer. Total

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proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Primary antibodies used were as follows: PPARγ (Abcam, Cambridge, UK), adiponectin (Abcam), and β-actin (Santa Cruz Biotechnology). The signals were

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quantified using the Fusion Solo Detector (Vilber Lourmat, Marne La Vallee, France).

2.7. Statistical analysis

The results are expressed as means ± SEM and were analysed using one-way ANOVA. Values of p < 0.05 were considered statistically significant.

3. Results 5

ACCEPTED MANUSCRIPT 3.1. Effects of hispidulin on 3T3-L1 preadipocyte viability We first confirmed the cytotoxicity of hispidulin to determine suitable doses for 3T3L1 preadipocytes. Treatments with hispidulin at concentrations of 0-40 µM had no

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significant effect on the viability of 3T3-L1 preadipocytes (Fig. 1). Thus, we used hispidulin at concentrations of 0-40 µM for subsequent experiments and also confirmed

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the non-cytotoxic effects of HCA.

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3.2. Effects of hispidulin on the differentiation of 3T3-L1 adipocytes To investigate the effects of hispidulin on the differentiation of 3T3-L1 preadipocytes into mature adipocytes, cells were treated with hispidulin at concentrations of 0-40 µM throughout the differentiation period. The morphology of the control cells changed to a

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more rounded shape and the cells accumulated lipid droplets, whereas the cells treated with hispidulin exhibited a preadipocyte-like morphology (Fig. 2A). In addition, the treatment with hispidulin decreased ORO staining and intracellular TG content in a

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concentration-dependent manner (Fig. 2B-D). When cells were treated with hispidulin at concentrations of 10, 20, and 40 µM, ORO–stained intracellular lipids were reduced

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to 67.4%, 62.4%, and 54.4% of the control values, respectively.Moreover, the antidifferentiation effect of hispidulin was higher than that of HCA at the same concentration.

3.3. Effects of hispidulin on mRNA and protein expression of adipogenesis-related 6

ACCEPTED MANUSCRIPT genes PPARγ and C/EBPα are major transcription factors of adipogenesis, and adiponectin is a protein secreted by adipocytes [10, 11]. To investigate the mechanisms of the anti-

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differentiation effects of hispidulin, we determined the expression of PPARγ, C/EBPα, and adiponectin. The treatment with hispidulin decreased the mRNA expression of PPARγ, C/EBPα, and adiponectin in a concentration-dependent manner (Fig. 3). When

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cells were treated with hispidulin at concentrations of 40 µM, the mRNA expression of PPARγ was reduced to 0.6-fold, compare to control. The protein expression of PPARγ

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and adiponectin was also reduced, similar to the mRNA expression pattern (Fig. 4).

4. Discussion

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In recent years, there has been a growing focus on the development of novel therapies for the treatment of obesity [12]. However, at the same time, several anti-obesity products have been withdrawn from the market after approval because of side effects

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[13]. For instance, sibutramine, a drug for long-term obesity treatment, was withdrawn in 2010 due to cardiovascular concerns, first in Europe, then in the US and Canadian

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markets [13]. Therefore, the safety of new therapeutic treatments must be carefully considered. In addition, strategies for the prevention of obesity using functional foods are considered important. Recently, many studies have demonstrated anti-obesity effects for natural compounds and plants as a dietary supplement or therapeutics [14-16]. For instance, the herbal mixture of polygala tenuifolia,Curcuma longa and Saururus chinensis exerted the anti-obesity activity in HFD-induced obese mice [14]. Also, the 7

ACCEPTED MANUSCRIPT bioactive compounds, such as rutin, quercitrin, and curcumin were detected in the herbal mixture [14]. Garcinia cambogia containing HCA has been widely marketed as a weight loss

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supplement worldwide [17-19]. In a previous study, the high dose of Garcinia cambogia containing HCA (154 mmol HCA/kg) was effective in suppressing epididymal fat accumulation in developing male Zucker obese rats [20].

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Hispidulin is found in several Artemisia, Salvia,and Scoparia species, and several Artemisia and Salvia species have been reported to help prevent obesity [21-23]. Many

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studies have reported the various pharmacological effects of hispidulin, such as antioxidant and antithrombotic effects [7, 24]. Previous studies demonstrated that hispidulin acts as a PPARγ and PPARα agonist in human embryonic kidney and hepatoma cell lines, respectively [23, 25]. Although these studies implied that hispidulin might also have a

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role in lipid storage and metabolism. They did not provide direct evidence for the antiadipogenic effects of hispidulin on adipocyte ; moreover , they were unable to confirm

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that PPARs are involved in not only energy homeostasis but also inflammaion [26]. In the present study, we therefore investigated the anti-adipogenic effects of hispidulin

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on adipocytes and confirmed the expression level of adiponectin, an adipokine secreted by adipocytes. Hispidulin at concentrations of 0-40 µM significantly inhibited the differentiation of 3T3-L1 adipocytes and lipid accumulation within them. These results suggest that the anti-obesity effect of S. plebeia extract (at doses of 0-400 mg/kg) in high-fat diet-induced obese mice, which was demonstrated in a previous study [6], probably caused by the hispidulin component of the extract. 8

ACCEPTED MANUSCRIPT A previous study showed the structure–activity relationships of chromone compounds with similar chemical structures; hispidulin has a similar chemical structure as some chromone derivatives (such as eupatilin and jaceosidin) [27]. In this regard, although we

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did not test this, we postulate that these compounds, with similar structures, may have similar effects.

We also compared the ant-differentiation effect of hispidulin with that of HCA.

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Hispidulin showed a significantly greater inhibitory effects on the differentiation and lipid accumulation of adipocytes than HCA at the same concentration. These results

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suggestthat hispidulin has superior anti-adipogenic effects for the prevention or treatment of obesity.

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5. Conclusion

Our findings are the first evidence, to our knowledge, to support the anti-adipogenic effects of hispidulin in 3T3-L1 adipocytes, possibly through the PPARγ pathway.

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Further research to elucidate the therapeutic role of hispidulin in vivo in an obesity

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animal model is warranted.

Conflicts of interest

The authors declare that there are no conflicts of interest.

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ACCEPTED MANUSCRIPT Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant

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funded by the Korea government (MEST) (No. NRF-2017R1A2B4011003).

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[27] H.-S. Kim, J.-H. Kim, J.Y. Lee, Y.-M. Yoon, I.-H. Kim, H.-S. Yoon, B.-S. Youn, Small molecule-mediated reprogramming of epithelial-mesenchymal transition thereby blocking

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fibrosis, bioRxiv, (2017).

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Hispidulin at concentrations of 0- 80 µM did not affect the viability of 3T3-L1 preadipocytes. Cell viability was confirmed by using the MTT assay, as

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described in the Materials and Methods section. Bars represent means ± SD from three independent experiments.

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Figure 2. Hispidulin inhibits the differentiation of 3T3-L1 cells into mature adipocytes. 3T3-L1 cells were treated with hispidulin at concentrations of 0- 40 µM

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throughout the differentiation period. (A) At the end of the treatment, the differentiated cells were photographed with a microscope at 200× magnification. (B) Cells were stained with Oil Red O (ORO) and photographed under a microscope at 200× magnification. (C) ORO stain in the cells was dissolved and measured. (D)

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Differentiated cells were lysed and intracellular triglyceride (TG) content was determined. Bars represent means ± SD from three independent experiments. P

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<0.01(**) and P <0.05(*).

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Figure 3. Hispidulin inhibits mRNA expression of adipogenesis-related genes. At the end of the treatment, total RNA was isolated and mRNA expression of PPARγ, C/EBPα, and adiponectin was evaluated by RT-qPCR, as described in the Materials and Methods section. The mRNA expression level of each gene was normalized to that of βactin and was expressed relative to the positive control (PC). The heatmap was generated from normalized data using Bio-Rad CFX Manager™ Software. Bars 13

ACCEPTED MANUSCRIPT represent means ± SD from three independent experiments. P <0.01(**) and P <0.05(*).

Figure 4. Hispidulin inhibits protein expression of adipogenesis-related genes. Total

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protein was isolated and protein expression of PPARγ, C/EBPα, and adiponectin was analysed by western blotting, as described in the Materials and Methods section. Protein

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expressed relative to the positive control (PC) values.

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expression levels were normalised to the corresponding β-actin levels and were

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Fig 1.

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100 80 60

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CON

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Cell viability (% of control)

120

10

20 Hispidulin (µM)

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40 HCA (µM)

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Fig. 2

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(A) HCA (µM)

20

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PC

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NC

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Hispidulin (µM)

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HCA (µM)

Hispidulin (µM)

(B) 20

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10

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PC

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(C)

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100 80 60 40

**

** **

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ORO staining (% of control)

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20 0 PC

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(D)

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100

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PC

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Triglyceride content (% of control)

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Fig. 3 1.4

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C/EBPα

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Fold change

PPARγ

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0.2 0 NC

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Fig. 4

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Hispidulin (µM) PPAR γ

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Adiponectin

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ACCEPTED MANUSCRIPT Highlights

Hispidulin inhibits the differentiation and lipid accumulation in 3T3-L1 adipocytes.

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Hispidulin suppresses the expression of adipogenic markers. Hispidulin can control obesity by regulating PPARγ pathway in adipocyte

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differentiation.